Mutations in the Microphthalmia (Mi) gene represent one of the oldest recognized causes of melanocyte death in mice (manifest as coat color abnormalities). Mi mutations have recently also been shown to occur in humans with the pigmentation disorder Waardenburg Syndrome Type 2. Mi is a factor whose function is clearly critical to melanocyte development. The Mi gene was recently cloned and encodes a factor with substantial homology to the Myc oncoprotein in a basic/helix-loop-helix/leucine zipper (b-HLH-ZlP) domain. Mi is one of the only proteins in this family which displays tissue specific expression. We have defined the DNA specificity of Mi and found it to bind and potently activate transcription off a promoter element previously shown to confer melanocyte specific expression of pigmentation genes. The conservation of this "M box" element in a number of melanocyte specific gene promoters, Mi's ability to activate it, and the devastating effects of Mi mutations on melanocyte survival strongly suggest that Mi is a master regulator for pigmentation and melanocyte development. We have also found three other b-HLH-ZIP factors with which Mi will heterodimerize (TFEB, TFEC, and TFE3) collectively called the "MiT" family. Despite its restricted expression, we have found Mi mRNA in all 13 melanomas examined to date, suggesting that like Myc its expression may modulate proliferation. We also found that the chromosomal map site for one of its dimerization partners (TFEB) is also frequently amplified in melanoma. Our characterization of 7 mutant alleles of Mi (each derived from a different mouse strain) revealed clustered mutations in or near the DNA binding domain. We found striking correlations between their inherited phenotypic severity and their biochemical behavior through structure/function studies. To further enhance our understanding of this factor and extend our analysis of its actions in melanocytes and melanoma, this grant proposes to : 1) examine Mi's protein:protein and protein: DNA recognition properties, 2) determine the transcriptional activities of Mi as well as its 3 binding partners we have identified, 3) analyze Mi expression using 51 protection, RT-PCR, and antibody directed protein analyses in melanocytes, and melanomas, 4) transfect wild type Mi and mutant which we have characterized into a variety of cells to study Mi's roles in melanocyte differentiated and proliferation.